EXPRESSION AND REGULATION OF THE HUMAN DOPAMINE TRANSPORTER IN A NEURONAL CELL-LINE

Human cocaine users exhibit increased striatal [H-3]WIN35428 binding t o the dopamine transporter (DAT). However, the nature of the changes i nduced in the DAT are complex and may not result from a simple increas e in number of DAT molecules. To better understand the regulation of D AT inhibitor binding sites and their relationship to the overall proce ss of dopamine uptake, a neuronal model system expressing the human DA T has been developed. Initial experiments were attempted with native d opaminergic neurons so as to allow examination of DAT interactions wit h vesicular release and storage mechanisms. Dissociated fetal rat mese ncephalic neurons, of various ages and mixtures with target cells, wer e grown to confluence. However, [H-3]WIN35428 binding was of low affin ity at all levels of maturity. Following this, a simpler model was ass essed, using DAT cDNA transfected into neuroblastoma-derived Neuro2A c ells. Initially, no specific and little non-specific [H-3]WIN35428 or [H-3]paroxetine binding was found in non-transfected cells. After tran sfection with the human DAT inserted in the pcDNA vector, both DAT bin ding and dopamine uptake were significantly and stably present. Treatm ent with (-)cocaine, 10(-6) M for 24 h, increased DAT binding and upta ke, which did not occur in parallel COS-7 experiments. Other experimen ts with Neuro2A cells also found that dopamine uptake was down-regulat ed by treatment with a PKC activator. These results suggest that the t ransfected Neuro2A neurons should be useful for ongoing experiments ex amining the regulation of the DAT by assorted treatments. (C) 1998 Els evier Science B.V. All rights reserved.